Application of pseudomonas aeruginosa vaccine in treating infection associated with burn or scald injury

ABSTRACT

The present invention belongs to the field of microbiology, and particularly relates to an application of a Pseudomonas aeruginosa vaccine in prevention and treatment of burn and scald complicated with bacterial infection. The burn and scald of the present invention include burns and scalds, and degree of the scalds includes I degree, superficial II degree, deep II degree, or III degree scalds. Site of the scalds includes skin, mucosa or other tissues. The Pseudomonas aeruginosa vaccine of the present invention can effectively prevent and treat burn and scald complicated with Pseudomonas aeruginosa infection caused by multidrug-resistant Pseudomonas aeruginosa by activating the specific immune response of the body. The Pseudomonas aeruginosa vaccine of the present invention can reduce the bacterial load in the immunized subject through the established immunization procedures, thereby providing a technical solution that can effectively prevent burn and scald complicated with Pseudomonas aeruginosa infection, which avoids the technical problems caused by the use of antibiotics such as poor effectiveness, difficulty in curing and proneness to drug resistance in the prior art to a certain degree.

PRIORITY APPLICATIONS

The present application claims priority from Chinese invention patentapplications 1) 201910777479.2 “BACTERIAL MEMBRANE VESICLE, ANDPREPARATION METHOD AND APPLICATION THEREOF”, 2) 201910777473.5“Staphylococcus aureus MEMBRANE VESICLE, AND PREPARATION METHOD ANDAPPLICATION THEREOF”, 3) 201910777606.9 “Pseudomonas aeruginosa MEMBRANEVESICLE, AND PREPARATION METHOD AND APPLICATION THEREOF”, 4)201921369450.2 “A PRODUCTION SYSTEM, AND ISOLATION AND PURIFICATIONSYSTEM FOR BACTERIAL MEMBRANE VESICLE”, 5) 201910777595.4 “A PRODUCTIONSYSTEM, AND ISOLATION AND PURIFICATION SYSTEM AND METHOD FOR BACTERIALMEMBRANE VESICLE” filed on Aug. 22, 2019, which are incorporated byreference in their entirety.

TECHNICAL FIELD

The present invention belongs to the field of microbiology, andparticularly relates to an application of a Pseudomonas aeruginosavaccine in prevention and treatment of burn and scald complicated withbacterial infection.

BACKGROUND

At present, burn and scald have become one of the most common accidentalinjuries in daily production and life. According to statistics, about5000-10000 people per million people in China suffer from burn or scaldevery year. The main parts of burn and scald include skin and/or mucosa,and in severe cases, subcutaneous and/or submucosal tissues, such asmuscles, bones, joints and even internal organs, can be injured. Scaldis a type of thermal burns, and is tissue damage mainly caused by hotfluid, steam and high-temperature solid. The depth of scald is judgedaccording to the classification rule of “three degrees and four types”for burn severity, and according to the depth, pathological change andclinical manifestations of scald, it can be divided into: I degree,superficial II degree, deep II degree and III degree scalds.

Wound infection is the most common and serious complication after scald.The bacteria which cause wound infection are mainly gram-negativebacilli, wherein Pseudomonas aeruginosa is one of the most commonpathogens. The detection rate of Pseudomonas aeruginosa in burn patientsvaries from 20% to more than 50% in different reports. Some studies havepointed out the probability that Pseudomonas aeruginosa could beisolated from burn wounds 7 days after burn was almost 100%. It is alsoreported that the mortality rate after Pseudomonas aeruginosa infectionis 31%, especially in patients with burn. If the bacterial infection ofburn wounds is not treated in time, it may cause serious consequences.In addition, if the bacteria in the burn wounds are not removed in time,delay of wound healing or healing with scar formation may impair thefunction of a wound site (joint).

The current treatment for scald complicated with Pseudomonas aeruginosainfection is generally surgical debridement combined with local andsystemic antibiotics. The most common antibacterial drugs includesynthetic antibacterial drugs such as silver sulfadiazine andantibiotics such as ciprofloxacin, ceftazidime andcefoperazone/sulbactam. However, due to the irrational use ofantibiotics and the emergence of multidrug-resistant bacteria, thetherapeutic effect is unsatisfactory, such as progression into sepsis,which is life-threatening. According to statistics, in the process ofscald complicated with Pseudomonas aeruginosa infection, Pseudomonasaeruginosa sepsis is the main reason of death in scald patients, with anincidence rate from 8% to 42.5% and a mortality rate from 28% to 65%.

SUMMARY

In view of this, the objective of the present invention is to provide anapplication of a Pseudomonas aeruginosa vaccine in prevention andtreatment of infection in burn and scald.

To achieve the above objective, the present invention adopts thefollowing technical solutions:

Use of a Pseudomonas aeruginosa vaccine in manufacture of a medicamentfor prevention and treatment of infection in burn and scald.

Further, the burn and scald include burns and scalds, and degree of thescalds includes I degree, superficial II degree, deep II degree, or IIIdegree scalds.

Further, site of the scalds includes skin, mucosa or other tissues.

Further, the infection in burn and scald is burn and scald complicatedwith bacterial infection.

Further, bacteria of the complicated with bacterial infection includeone or more of Pseudomonas aeruginosa, Klebsiella pneumoniae,Acinetobacter baumannii, Escherichia coli, Staphylococcus aureus,Streptococcus pneumoniae and Mycobacterium tuberculosis.

Further, the bacterial infection is Pseudomonas aeruginosa infection.

Further, the burn and scald include burns and scalds, and degree of thescalds includes I degree, superficial II degree, deep II degree, or IIIdegree scalds.

Further, site of the scalds includes skin, mucosa or other tissues.

Further, the Pseudomonas aeruginosa vaccine comprises inactivatedPseudomonas aeruginosa and/or Pseudomonas aeruginosa membrane vesicles.

Further, immunization procedures of the Pseudomonas aeruginosa vaccinecomprise: injection take places (i) 0, 3rd, and 7th days, and (ii) 0,2nd and 4th weeks.

Further, the inactivated Pseudomonas aeruginosa is inactivated byirradiation, and the Pseudomonas aeruginosa membrane vesicles areisolated from the Pseudomonas aeruginosa inactivated by irradiation.

Further, the Pseudomonas aeruginosa vaccine prevents Pseudomonasaeruginosa infection, and reduces bacterial load in skin scaldcomplicated with bacterial infection.

Further, content of whole-cell Pseudomonas aeruginosa in the Pseudomonasaeruginosa vaccine comprises: 1×10⁴-1×10¹⁰/injection.

Further, the content of whole-cell Pseudomonas aeruginosa in thePseudomonas aeruginosa vaccine comprises: 1×10⁴/injection,1×10⁵/injection, 1×10⁶/injection, 1×10⁷/injection, 1×10⁸/injection,1×10⁹/injection and 1×10¹⁰/injection.

Further, the Pseudomonas aeruginosa vaccine further contains animmunoadjuvant.

Further, administration site of the Pseudomonas aeruginosa vaccine issubcutaneous, muscle and/or mucosa.

Further, the medicament can also contain any pharmaceutically acceptablecarrier and/or adjuvant.

Further, the carrier is a liposome.

The present invention has the following beneficial effects:

The experimental results of the present invention show that thePseudomonas aeruginosa vaccine of the present invention can effectivelyprevent and treat burn and scald complicated with Pseudomonas aeruginosainfection caused by multidrug-resistant Pseudomonas aeruginosa byactivating the specific immune response of the body. The Pseudomonasaeruginosa vaccine of the present invention can reduce the bacterialload in the immunized subject through the established immunizationprocedures, thereby providing a technical solution that can effectivelyprevent burn and scald complicated with Pseudomonas aeruginosainfection, which avoids the technical problems caused by the use ofantibiotics such as poor effectiveness, difficulty in curing andproneness to drug resistance in the prior art to a certain degree.

The experimental results of the present invention also show that, thePseudomonas aeruginosa vaccine of the present invention can be used toeffectively inhibit the bacterial load in the site of burn and scaldcomplicated with bacterial infection by activating the human immuneresponse, prevent secondary infection caused by Pseudomonas aeruginosaand avoid the problem of drug resistance caused by antibiotics in theprior art, and has broad application scenarios in burn and scaldcomplicated with bacterial infection.

DESCRIPTION OF DRAWINGS

To make the embodiments of the present invention or the technicalsolutions in the prior art clearer, the drawings required to be used inthe description of the embodiments or the prior art will be brieflyintroduced below. It is obvious that the drawings described below aresome embodiments of the present invention, and that other drawings canbe obtained from these drawings for those of ordinary skill in the artwithout making inventive effort.

FIG. 1 is a Transmission Electron Microscopy (TEM) image of irradiatedPseudomonas aeruginosa membrane vesicles (scale: 200 nm).

FIG. 2 shows the percentage of proliferation of CD4⁺ T cells afterinteracting with DC treated with different treatment methods.

FIG. 3 is a flow cytometry plot of proliferation of CD4⁺ T cells afterinteracting with DC treated with different treatment methods.

FIG. 4 shows irradiated membrane vesicles enhance the interactionbetween DC cells and T cells (GC: growth control, dendritic cell growthcontrol group (unstimulated group); Cell+MVs (whole-cellbacteria+membrane vesicle treatment group); MVs (membrane vesicletreatment group)).

FIG. 5 shows the bacterial load of scalded sites of rabbits 24 h afterPseudomonas aeruginosa infection.

FIG. 6 shows the bacterial load of scalded sites of rabbits 24 h afterPseudomonas aeruginosa PA14 infection.

DETAILED DESCRIPTION

To make the objective, the technical solutions and advantages of theembodiments of the present invention clearer, the technical solutions ofthe embodiments of the present invention will be clearly and completelydescribed below in combination with drawings. It is obvious that thedescribed embodiments are some of the embodiments of the presentinvention, not all of the embodiments. Based on the embodiments of thepresent invention, all other embodiments obtained by those of ordinaryskill in the art without making inventive effort shall belong to theprotection scope of the present invention.

The term “burn and scald” in the present invention refers to the damageto tissues, mainly referring to skin and/or mucosa, caused by heat,including hot fluid (water, soup, oil, etc.), steam, high-temperaturegas, flame, and red-hot metal liquid or solid (such as molten steel andsteel ingots), which may damage subcutaneous and/or submucosal tissuesin severe cases, such as muscles, bones, joints and even internalorgans. Scald is tissue damage caused by hot fluid, steam andhigh-temperature solid, and belongs to one of thermal burns.

The Pseudomonas aeruginosa vaccine of the present invention comprises(i) irradiation-inactivated Pseudomonas aeruginosa cells and/or (ii)Pseudomonas aeruginosa membrane vesicles. FIG. 1 is a TransmissionElectron Microscopy (TEM) photograph of purified membrane vesicles.

Embodiments 1-3 introduce some isolation methods for preparing vesicles.The vesicles can be isolated from non-irradiated bacteria or isolatedfrom irradiated bacteria or obtained by other methods.

Embodiment 1 Isolation Method for Bacterial Membrane Vesicles

In some embodiments, a method for isolating membrane vesicles fromPseudomonas aeruginosa comprises the following steps: 1) isolatingbacterial cells in bacterial solution for culture of bacteria fromculture medium, and collecting supernatant 1; 2) centrifuging thesupernatant 1 with a high-speed centrifuge, and collecting supernatant2; and 3) centrifuging the supernatant 2 with an ultra-high-speedcentrifuge to precipitate membrane vesicles.

Further, isolation method in the step 1) comprises centrifugation,column chromatography, or dialysis bag concentration.

Further, the supernatant 2 collected in the step 2) is subjected todialysis bag concentration prior to the step 3). In some embodiments,the dialysis bag selected can concentrate substances greater than 100KD.

Further, the membrane vesicles are resuspended with a buffer solution,the buffer solution comprises 50 mM Tris, 5 mM NaCl and 1 mM MgSO₄calculated as a volume unit of 1 L and has a pH of 7.4.

In some embodiments, the bacterial cells and the membrane vesicles areprepared as a biological composition, and the preparation methodcomprises: collecting the bacterial cells isolated in the step 1) in theabove isolation method for membrane vesicles, and mixing the bacterialcells with the membrane vesicles obtained in the step 3) to form thebiological composition.

Further, in the step 1), the supernatant 1 is filtered with a 0.3-0.5 μMfilter to remove impurities.

Preferably, the supernatant 1 is filtered with a 0.45 μM filter toremove impurities.

Further, the isolation method in the step 1) is centrifugation, thecentrifugation speed is 100-10000 g, and the centrifugation time is10-60 min.

Preferably, the centrifugation speed in the step 1) is 400-8000 g, andthe centrifugation time is 10-30 min.

Further, the high-speed centrifugation speed in the step 2) is5000-25000 g, and the high-speed centrifugation time is 10-100 min.

Preferably, the high-speed centrifugation speed in the step 2) is10000-20000 g, and the high-speed centrifugation time is 30-60 min.

Further, the ultra-high-speed centrifugation speed in the step 3) is5000-150000 g, and the ultra-high-speed centrifugation time is 60-600min.

Preferably, the ultra-high-speed centrifugation speed in the step 3) is15000-150000 g, and the ultra-high-speed centrifugation time is 60-180min.

Embodiment 2 Augmentation and Purification of Bacterial MembraneVesicles

Further, in some embodiments, the method for preparing bacterialmembrane vesicles further comprises the following steps: 1) Augmentationof membrane vesicles: culturing bacteria to logarithmic growth phase;collecting the bacterial cells, resuspending the bacterial cells andthen irradiating them with ionizing irradiation to obtain irradiatedbacteria; 2) Isolation and purification of membrane vesicles: isolatingmembrane vesicles produced by the irradiated bacteria from theirradiated bacteria to obtain the membrane vesicles using the method forisolating membrane vesicles described in Embodiment 1.

Further, the ionizing irradiation is X-rays, and the irradiation dose is500-3000 Gy. The irradiation dose specifically comprises: 500-600 Gy,600-700 Gy, 700-800 Gy, 800-900 Gy, 900-1000 Gy, 1000-1100 Gy, 1100-1200Gy, 1200-1300 Gy, 1300-1400 Gy, 1400-1500 Gy, 1500-1600 Gy, 1600-1700Gy, 1700-1800 Gy, 1800-1900 Gy, 1900-2000 Gy, 2100-2200 Gy, 2200-2300Gy, 2300-2400 Gy, 2400-2500 Gy, 2500-2600 Gy, 2600-2700 Gy, 2700-2800Gy, 2800-2900 Gy and 2900-3000 Gy.

Preferably, the irradiation dose is 500-1000 Gy. The irradiation dosespecifically comprises: 500-600 Gy, 600-700 Gy, 700-800 Gy, 800-900 Gyand 900-1000 Gy.

Further, OD₆₀₀ value of the bacteria in logarithmic growth phase in thestep 1) is 0.3-0.8.

Preferably, the OD₆₀₀ value of the bacteria in logarithmic growth phasein the step 1) is 0.5-0.7.

Further, in the step 1), the bacterial cells are resuspended withphosphate buffer saline or sterile normal saline.

Preferably, in the step 1), the bacterial cells are resuspended withphosphate buffer saline.

Further, in the step 1), the bacterial cells are resuspended to an OD₆₀₀value of 20-80.

Preferably, in the step 1), the bacterial cells are resuspended to anOD₆₀₀ value of 40-60.

The content of nucleic acids and the content of proteins in the membranevesicles prepared by the above method are increased by 10-20 times,compared with those prepared from bacteria not irradiated with ionizingirradiation.

The membrane vesicles prepared by the present invention have variousapplication scenarios: for example, (i) the membrane vesicles can beused as an immunogen; (ii) the membrane vesicles can be used as animmune response enhancer; (iii) the membrane vesicles can be used as avaccine for treating bacterial infectious diseases; (iv) the membranevesicles can be used as a vaccine adjuvant (in some embodiments, thevaccine adjuvant non-specifically changes or enhances theantigen-specific immune response of the body); (v) the membrane vesiclescan be used as an antigen-presenting cell function enhancer.

The above antigen-presenting cell includes dendritic cells (i.e., DCcells), macrophages and B cells. The membrane vesicles obtained byirradiation, isolation and purification can be used as an enhancer forthe maturation of the DC cells, and specifically, used as an enhancerfor promoting the significant up-regulation of cell surface moleculesCD80, CD86 and MHCII molecules of bone marrow-derived dendritic cells.

In some embodiments, the membrane vesicles prepared by the presentinvention can be combined with DC cells in preparation of aproliferation agent for CD4⁺ T cells. Specifically, the method forpromoting proliferation of CD4⁺ T cells comprises the following steps:co-culturing membrane vesicles-stimulated and OVA-antigen-phagocytosedDCs with CFSE-labeled CD4⁺ T lymphocytes in vitro, wherein the membranevesicles are prepared by irradiation.

Embodiment 3 A Method for Isolating and Preparing Bacterial MembraneVesicles

In some embodiments, the method for isolating and preparing bacterialmembrane vesicles comprises the following steps:

1. Culturing bacteria to logarithmic growth phase, wherein OD₆₀₀ valueof the bacteria in logarithmic growth phase is 0.3-0.8, and the OD₆₀₀value of 0.5-0.8 is preferably selected (fermentation can also beperformed here to further enrich bacterial cells); collecting bacterialcells, and resuspending the bacterial cells with an appropriate amountof phosphate buffer solution, wherein the ratio of the amount of theadded phosphate buffer solution to the total amount of the bacterialcells is that the OD₆₀₀ value of the amount of the bacteria contained inevery 1 ml of solution is 20-80, and the OD₆₀₀ value of 40-60 ispreferably selected; after resuspension, irradiating the bacterial cellswith ionizing irradiation to obtain irradiated bacteria; preferably,irradiating with X-rays, with an irradiation dose of 500-3000 Gy. Theirradiation dose specifically comprises: 500-600 Gy, 600-700 Gy, 700-800Gy, 800-900 Gy, 900-1000 Gy, 1000-1100 Gy, 1100-1200 Gy, 1200-1300 Gy,1300-1400 Gy, 1400-1500 Gy, 1500-1600 Gy, 1600-1700 Gy, 1700-1800 Gy,1800-1900 Gy, 1900-2000 Gy, 2100-2200 Gy, 2200-2300 Gy, 2300-2400 Gy,2400-2500 Gy, 2500-2600 Gy, 2600-2700 Gy, 2700-2800 Gy, 2800-2900 Gy and2900-3000 Gy.

2. Collecting bacterial solution, centrifuging the bacterial solutionand collecting supernatant, and filtering the supernatant with a 0.3-0.5μM filter to remove the bacteria; wherein the centrifugation speed is400-8000 g; and the centrifugation time is 10-30 min.

3. Centrifuging the filtered supernatant with a high-speed centrifuge,collecting supernatant, and removing flagella; wherein the high-speedcentrifugation speed is 10000-20000 g; and the high-speed centrifugationtime is 30-60 min.

4. Centrifuging the supernatant after removal of the flagella with anultra-high-speed centrifuge to precipitate membrane vesicles; whereinthe ultra-high-speed centrifugation speed is 15000-150000 g; and theultra-high-speed centrifugation time is 60-180 min.

5. Collecting the membrane vesicles to obtain purified membranevesicles.

Preparation, isolation and purification of membrane vesicles byirradiating Pseudomonas aeruginosa PAO1 with ionizing irradiation:

1. Streaking Pseudomonas aeruginosa PAO1 recovered from −80° C. onto LBplates, and culturing them in an incubator at 37° C. for 16-18 h.

2. Picking monoclonal colonies from the LB plates, inoculating themonoclonal colonies in 20 mL of LB liquid medium, and culturing them atconstant temperature of 37° C. at 250 rpm for 16-18 h.

3. Inoculating overnight bacterial solution into 1 L of LB medium to aninitial concentration of 0.05 OD₆₀₀/mL and culturing the bacteria tologarithmic growth phase at 37° C. at 250 rpm, and measuring OD₆₀₀ valueof the bacterial solution.

4. Transferring the above bacterial solution of the step 3 to acentrifugal barrel, centrifuging the bacterial solution at 5,000 g for20 min, collecting the bacterial cells and resuspending the bacterialcells with normal saline, and adjusting the concentration of thebacterial cells to about 50 OD.

5. Placing the above bacterial solution in an irradiator with anirradiation dose of 1000 Gy.

6. Centrifuging the irradiated bacterial solution at 8,000×g for 20 mintwice and collecting supernatant; filtering the supernatant with a 0.45μM filter to remove the bacteria and collecting the supernatant again;at the same time, coating a small amount of the supernatant onto the LBplates and culturing them at 37° C. for 24-72 h to confirm that viablebacteria do not exist.

7. Centrifuging the supernatant of the step 6 with a high-speedcentrifuge to remove flagella in the supernatant.

8. Centrifuging the supernatant of the step 7 with an ultra-high-speedcentrifuge to precipitate membrane vesicles.

9. Discarding the supernatant, resuspending the precipitate with MVbuffer, and storing it at −80° C.

10. Observing the extracted membrane vesicles of the normal group andthe membrane vesicles of the experimental group of the present inventionby transmission electron microscopy.

Experimental Results:

According to the results of transmission electron microscopy, theionizing irradiation can stimulate Pseudomonas aeruginosa PAO1 toproduce membrane vesicles. The membrane vesicles are shown in FIG. 1.

Embodiment 4 Immunomodulatory Effects of Irradiated Bacterial MembraneVesicles—Promoting Maturation of Dendritic Cells

Dendritic cells (DCs) are the main antigen-presenting cells of the body,and have the main function of phagocytosing and processing antigenmolecules as well as presenting them to T cells. The DCs are the knownmost powerful and the only professional antigen-presenting cell that canactivate resting T cells in the body, and are a key link in initiating,regulating and maintaining immune responses. The maturation of the DCsdetermines the immune response or immune tolerance of the body.Co-stimulatory molecules B7 (B7-1=CD80 and B7-2=CD86) on the surfaces ofthe DCs can be bound to CD28 or CD152 molecules on the surfaces of Tcells, to enhance or weaken the MHC-TCR signal transduction between DCsand T cells. The main characteristics of mature DCs are changes in theexpression of co-stimulatory molecules CD80 and CD86, reduced ability tophagocytose antigens and enhanced the ability to process and presentantigens (increased MHCII molecules expression), and interaction with Tlymphocytes.

1. Culture and induction of mouse bone marrow-derived dendritic cells(BMDC): taking 6-8 week old C57 female mice, aseptically separatingmouse femurs, removing the muscles on the femurs, and cutting both endsof the femurs; rinsing the bone lumens with PBS until the bone lumensturn white; filtering PBS suspension and then centrifuging it at 1200rpm for 5 min; removing supernatant; and adding 5 ml of red blood celllysis buffer to resuspend the cells. After standing for 15 min,centrifuging the lysis product at 1200 rpm for 5 min, and removing thesupernatant; adding 50 ml of 1640 complete medium (20 ng/ml GM-CSF, 10%FBS and 50 mM of 2-mercaptoethanol) to resuspend the cells. Afteruniform mixing, dividing the cells into 5 petri dishes and culturingthem in an incubator. Changing the medium every 2 days and collectingthe cells on the 7th day.

2. BMDC stimulation: taking the BMDC cells induced for 7 days, andrepeatedly blowing the cells in a 6-well plate to detach adherent cells;collecting the cell suspension, centrifuging it at 1100 rpm for 5 min,removing supernatant, and adding 1 ml of medium to resuspend the cells,and adjusting the cell concentration to 1×10⁶/m1 after counting viablecells, and inoculating 2 ml of the cells into a new 6-well plate. Eachstimulator will be added respectively and uniformly mixed: whole-cellbacteria, whole-cell bacteria+vesicles, and vesicles at a finalconcentration of 15 μg/mL (based on protein). Continuing to culture themfor 24 hours and adding an equal volume of PBS to the growth controlgroup.

3. Maturation markers detection by flow cytometry: after 24 h, takingout the 6-well plate, repeatedly blowing the cells to detach them,collecting the cell suspension into a Flow Cytometry Tube, centrifugingit at 1500 rpm for 3 min, removing supernatant, adding 1 ml of PBS tocontinue centrifugation at 1500 rpm for 3 min, then removing thesupernatant and repeatedly washing for 3 times. AddingCD11c/CD80/CD86/MHCII antibodies and incubating at room temperature for30 min in the dark; at the same time, setting an isotype control groupas the negative control group (adding isotype controls ofCD11c/CD80/CD86/MHCII). After incubation, adding PBS to wash twice, thenadding 200 μl of PBS to resuspend the cells, and detecting the cells byflow cytometry.

4. Result processing: analyzing the ratio of CD80/CD86/MHCII in CD11ccells by flow cytometry software.

Experimental results: Compared with the whole-cell bacteria, thevesicles of the experimental group (MVs) treated by X-rays cansignificantly up-regulate the surface costimulatory molecules CD80, CD86and MHCII of DCs after stimulation, and these surface molecules aremarkers of dendritic cell maturation. In conclusion, it is proved thatthe vesicles can significantly promote the differentiation andmaturation of DCs.

The phagocytic ability of DC cells is detected by detecting thefluorescence intensity of FITC-dextran: DC cells have strong antigenendocytosis and processing abilities. The DC cells have strongphagocytic ability in an immature state when not in contact withantigen. After in contact with antigen and activated, the DC cellsbecome mature with low phagocytic ability and enhancedantigen-presenting ability. In the experiment, by detecting thefluorescence intensity of the FITC-dextran, the amount of the dextranphagocytosed by DC is determined to detect whether the phagocyticability of DC is enhanced.

1. Culture and induction of BMDC cells (same as the above).

2. Stimulation: collecting the cells on the 7th day, blowing down allthe cells, then centrifuging and resuspending the cells for counting,then inoculating the cells into a 6-well plate with 1×10⁶ cells perwell, and respectively adding the stimulator: adding an equal volume ofPBS to the GC group, adding the same concentration of membrane vesicles(by protein level) to the control group and the treatment group and thenculturing them at 37° C. for 24 h.

3. Phagocytosis and detection: adding the dextran (5 μg/ml), and afterculture for 1 h, aspirating the cells into a Flow Cytometry Tube;washing the cells with PBS for 3 times; adding CD11c antibody andincubating at room temperature for 30 min in the dark; washing the cellswith PBS for 3 times; and detecting the fluorescence of FITC by flowcytometry.

4. Result processing: analyzing the ratio of FITC in CD11c cells by flowcytometry software.

Experimental results: in order to detect the phagocytic function of DCs,the present invention uses FITC-dextran as a model antigen forphagocytosis of DCs and detects the mean fluorescence intensity value ofFITC of CD11c+DCs. The experimental result shows that after the DCs arestimulated by membrane vesicles, the mean fluorescence intensity valueof FITC is significantly reduced compared with that of the GC group(growth control group). This experimental result proves again that thevesicles can promote the maturation of DCs, thereby reducing theirability to take up antigen.

Embodiment 5 Interaction Between Mature DCs and T Cells Stimulated byBacterial Membrane Vesicles in X-Ray Treatment Group

A. Interaction Between Mature DCs and CD4+ T Cells:

The effective cross-antigen presentation of extracellular proteins byDCs plays an important role in the induction of specific cellular immuneresponses. Therefore, the cross-presentation effect of OVA antigen byDCs stimulated by membrane vesicles is detected. 72 h after co-cultureof DCs-T cells, the proliferation of OT-II CD4⁺ T lymphocytes isdetected by CFSE flow cytometry. Fluorescent dye CFSE (CFDA-SE), namelycarboxyfluorescein diacetate, succinimidyl ester, is a cell stainingreagent that can fluorescently label live cells. CFDA-SE can beirreversibly coupled to cellular proteins by binding to intracellularamines after entering cells. In the process of cell division andproliferation, the CFSE-labeled fluorescence can be equally distributedto two daughter cells, and the fluorescence intensity is half that ofthe parental cells. Therefore, the percentage of cells with weak CFSEfluorescence can be counted by flow cytometry to obtain the proportionof proliferating cells.

1. Culture and induction of BMDC cells (same as the previousembodiment).

2. Antigen phagocytosis: culturing DCs which are cultured for 7 days ina medium containing 10 μg/ml OVA for 24 h to serve as GC (growth controlgroup); adding vesicles to the MVs group, then centrifuging andcollecting antigen-phagocytosed DCs; resuspending the DCs in a normalmedium; and applying the DCs in a 96-well plate at a density of 2×10⁴cells/well, with 100 μl per well, and 3 replicate wells per group.

3. T cell extraction: on the second day, isolating and enrichingOVA-specific CD4+T lymphocytes from the spleens of OT-II mice by amagnetic negative selection beads kit from Stem Cell Technologiescompany.

4. Co-culture of DC and T cells: labeling the sorted CD4⁺ T cells with 1μM CFSE according to the kit instructions. After labeling, washing thecells for 3 times with PBS and adding the cells to the 96-well plate ata density of 10⁵ cells/well to a final culture volume of 200 μl(CD4:DC=5:1).

5. On the 3rd day after co-culture, detecting the proliferation of CD4+T cell population by CFSE decrement by flow cytometry.

Experimental results: co-culturing membrane vesicles-stimulated andOVA-antigen-phagocytosed DCs with CFSE-labeled OT-II mouse CD4⁺ Tlymphocytes in vitro. The analysis results of flow cytometry for CFSEfluorescence intensity show that the proportion of proliferating CD4+ Tcells is increased. The membrane vesicles (14.05%) can significantlyincrease the proliferation-promoting effect of OVA-antigen-phagocytosedDCs (6.80%) on specific CD4⁺ T cells. See FIG. 2 and FIG. 3 for details.

B. Promotion of T Cell Proliferation by DCs Treated by the MembraneVesicles:

The effective cross-antigen presentation of extracellular proteins byDCs plays an important role in the induction of specific cellular immuneresponses. Therefore, the cross-presentation effect of OVA antigen byDCs stimulated by membrane vesicles is detected. 72 h after co-cultureof DCs-T cells, the proliferation of T lymphocytes is detected by CFSEby flow cytometry. Fluorescent dye CFSE (CFDA-SE), namelycarboxyfluorescein diacetate, succinimidyl ester, is a cell stainingreagent that can fluorescently label live cells. CFDA-SE can beirreversibly coupled to cellular proteins by binding to intracellularamines after entering cells. In the process of cell division andproliferation, the CFSE-labeled fluorescence can be equally distributedto two daughter cells, and the fluorescence intensity is half that ofthe parental cells. Therefore, the percentage of cells with weak CFSEfluorescence can be counted by flow cytometry to obtain the proportionof proliferating cells.

1. Culture and induction of BMDC cells (same as the previousembodiment).

2. Antigen phagocytosis: culturing DCs which are cultured for 7 days ina medium for 24 h to serve as GC (growth control group); adding vesiclesto the MVs group, then centrifuging and collecting antigen-phagocytosedDCs; resuspending the DCs in a normal medium; and applying the DCs in a96-well plate at a density of 4×10⁴ cells/well, with 100 μl per well,and 3 replicate wells per group.

3. T cell extraction: on the second day, isolating and enriching the Tcells of the mice from the spleens of mice one week after one MVsimmunization using a magnetic negative selection beads kit from StemCell Technologies company.

4. Co-culture of DC and T cells: labeling the sorted T cells with 1 μMCFSE according to the kit instructions. After labeling, washing thecells for 3 times with PBS and adding the cells to the 96-well plate ata density of 4×10⁵ cells/well to a final culture volume of 200 μl(CD3:DC=10:1).

5. On the 3rd day after co-culture, detecting the proliferation of CD3⁺,CD8⁺ and CD4⁺ T cell populations by CFSE decrement by flow cytometry.

Experimental results: co-culturing membrane vesicles-stimulated andOVA-antigen-phagocytosed DCs with CFSE-labeled OT-II mouse CD4⁺ Tlymphocytes in vitro. The analysis results of flow cytometry for CFSEfluorescence intensity show that the proportion of proliferating CD4⁺ Tcells is increased. As shown in the figure, the fluorescence intensityof the whole-cell bacteria plus vesicle stimulation group is 63.5%, andthe fluorescence intensity of the vesicle stimulation group is 71%. Itindicates that DCs after vesicle treatment can significantly stimulatethe proliferation of CD4⁺ T cells. See FIG. 4.

Embodiment 6 Experiment of Pseudomonas aeruginosa Vaccine Against ScaldComplicated with Pseudomonas aeruginosa Skin Infection

Experimental rabbits are used in the experiment. A scald model ofrabbits is established by scalding rabbits with a hot-air gun one daybefore the rabbits are infected with Pseudomonas aeruginosa. After 24 h,the scalded sites are subcutaneously infected with the homologous strainof Pseudomonas aeruginosa SKLBPA1 and Pseudomonas aeruginosa PA14respectively (both purchased from ATCC). 24 h after infection, therabbits are sacrificed, and skin and subcutaneous muscle tissue of thescalded sites are taken, homogenized and counted for CFU. The CFU of themodel group and the vaccine group are compared to observe whether thevaccine of the present invention has a protective effect on scaldcomplicated with Pseudomonas aeruginosa infection. The results of theexperiment will show that the Pseudomonas aeruginosa vaccine provided bythe present invention can have preventive and therapeutic effects onburn and scald complicated with infection caused by different serotypesof Pseudomonas aeruginosa (two representative strains of SKLBPA1 andPA14 are used as examples in the experiment), and has wide applicationscenarios.

The content of the vaccine of the present invention includes: 10⁸ CFU/ml

Experimental animals: 6 rabbits, New Zealand White rabbits, weighing2.210 kg-2.870 kg, female.

1. Experimental Groups

Three groups are set in the experiment with 2 animals in each group. Thespecific group information is shown in Table 1.

TABLE 1 Group Information Group Immunization Bacteria for Number GroupName Vaccine Procedure Infection Group PA1 PA1 0, 3, 7 d (injectionSKLBPA1 1 immunization take places at 0, group 1 3rd, 7th days) GroupPA1 0, 2, 4 w (injection 2 immunization take places at 0, group 2 2nd,4th weeks) Group PA1 model — — 3 group

2. Immunization

The test vaccine (10⁸ CFU/ml) is subcutaneously immunized with 1000 inthe left and right groins of the rabbits for 3 times, with twoimmunization procedures of 0, 3, 7 d (injection take places at 0, 3rd,and 7th days) and 0, 2, 4 w (injection take places at 0, 2nd and 4thweeks).

3. Establishment of Scald Model

Two days before infection, the front and back hairs on the left andright sides of the rabbits are removed by scissors and depilatory cream.After 24 h, the skin of the depilation sites is disinfected by 75%alcohol and applied with tetracaine hydrochloride mucilage for localanesthesia. Then, the temperature of the hot-air gun is set at 200° C.;the metal plate with square holes is disinfected by 75% alcohol andstuck onto the depilation sites. The hot-air gun is turned on; and a hotair outlet is pointed towards the square holes to blow the hot air ontothe rabbit skin for 5 s, causing a certain degree of scald model.

4. Infection

4.1 Recovery of Bacteria

Pseudomonas aeruginosa SKLBPA1 and PA1 recovered from −80° C. to TSAplates, and cultured overnight at 37° C.

4.2 Overnight Bacteria Culture in a Shaker

Monoclonal colonies are picked from the plates respectively into TSB forshaking culture at 37° C. at 220 rpm overnight.

4.3 Culture Expansion

The overnight bacterial solution is diluted to measure OD₆₀₀; theoriginal bacterial solution is inoculated into 100 ml of TSB (250 mlconical flask); and the bacteria are shaken at 37° C. at 220 rpm tologarithmic growth phase.

4.4 Centrifugation and Washing

The bacterial solution is collected into a 50 ml centrifuge tube, andcentrifuged at 4100 rpm (3000×g) at room temperature for 10 min; thesupernatant is discarded and the precipitate is resuspended with 2 mL of0.9% sodium chloride injection (about 5 OD/ml); and the bacterialsolution is adjusted to 0.1 OD₆₀₀ (2×10⁷ CFU/ml).

4.5 Infection

One week (the 7th day) after the last immunization, 50 μl of bacterialsolution (1×10⁶ CFU/site) is subcutaneously injected into the scaldedskin of the rabbits.

4.6 Plate Coating and Counting

The adjusted bacterial solution is coated to a TSA plate, and culturedat 37° C. overnight, and CFU is counted.

5. Count of Bacteria of Skin and Muscular Tissue 24 Hours afterInfection

Animals are sacrificed 24 hours after infection. The skin is disinfectedby spraying with 75% alcohol. The skin and muscular tissue of thescalded sites are taken aseptically, and the tissue is homogenized,coated to the TSA plate, and cultured at 37° C. overnight. The CFU iscounted by a colony counter.

6. Data Processing

LOG₁₀ CFU scatter plots of skin and muscular tissue are drawn usingGraphpad Prism software. The mean value of LOG₁₀ CFU is calculated, andbetween-group variation is analyzed by Analysis of Variance.

Experimental Results:

1. Change in Body Weights of Rabbits

The body weights of the animals in each group are shown in Table 2. Thebody weights of the rabbits in each immunization group and each modelgroup are increased steadily, and there is no significant differenceamong the groups.

TABLE 2 Change in Body Weights of Rabbits Week 1 Week 2 Week 3 Week 4Week 5 Week 6 Groups weight (kg) weight (kg) weight (kg) weight (kg)weight (kg) weight (kg) Group 1 2.460 ± 0.057 2.540 ± 0.057 2.590 ±0.028 2.655 ± 0.035 2.740 ± 0.085 2.905 ± 0.205 Group 2 2.540 ± 0.1982.615 ± 0.177 2.700 ± 0.170 2.770 ± 0.184 2.925 ± 0.148 3.025 ± 0.021Group 3 2.555 ± 0.064 2.635 ± 0.064 2.735 ± 0.078 2.800 ± 0.071 2.845 ±0.021 3.060 ± 0.156

2. Modeling Results of Skin Scald of Rabbits

The hot-air gun is sleeved with an air nozzle with an outer diameter of14 mm and an inner diameter of 10 mm; the temperature is set as 200° C.;and the air nozzle is kept close to the depilation sites on the backs ofthe rabbits for 5 seconds. After 2 hours, round blisters with a diameterof about 10 mm appear on the skins of the scalded sites, and after 24hours, the skins of the scalded sites are scabbed.

3. Infective Dose of Pseudomonas aeruginosa for Rabbit Skin

The concentrations of Pseudomonas aeruginosa SKLBPA1 and PA14 bacterialsuspensions used in the experiment are 0.1 OD/ml; the remainingbacterial suspensions after subcutaneous infection are diluted to 10⁻⁴and 10⁻⁵; 50 μl of bacterial suspensions are evenly coated on the TSAplates, and incubated overnight in an incubator at 37° C.; and the CFUis counted. The results are shown in Table 3.

TABLE 3 Viable Count of Bacteria Results of Bacterial Solution forInfection Turbidity of Concentration of Bacterial Strain BacterialSolution Bacterial Solution Name for Infection (OD/ml) for Infection(CFU/ml) SKLBPA1 0.1 8.00 × 10⁷ PA14 0.1 1.06 × 10⁸

4. Bacterial Load of Scalded Sites of Rabbits 24 h after Pseudomonasaeruginosa Infection

The animals are sacrificed 24 h after infection. The skin and musculartissue of the scalded and infected sites (infected with Pseudomonasaeruginosa SKLBPA1) are taken aseptically, homogenized, and coated tothe TSA plates, and cultured overnight at 37° C. The CFU is counted, andthen the mean and the standard deviation of each group are comparedbased on the log values of the bacterial loads to the base of 10. Theresults are shown in Table 4 and FIG. 5. Two immunization procedures ofPA1 vaccine can significantly reduce the load of Pseudomonas aeruginosaSKLBPA1 (P<0.01), which indicates that the vaccine has an obviousprotective effect.

TABLE 4 Bacterial Load of Scalded Sites of Rabbits 24 h AfterPseudomonas Aeruginosa Infection Number Bacteria for of Skins LOG10 ofGroups Vaccine Immunization Procedure Infection (blocks) Bacterial LoadGroup 1 PA1 0, 3, 7 d (injection take places SKLBPA1 6 5.08 ± 0.61** at0, 3rd, 7th days) Group 2 0, 2, 4 w (injection take places 6 4.68 ±0.40** at 0, 2nd, 4th weeks) Group 3 — — 6 6.93 ± 0.57  (Note:**compared with each model group, P < 0.01 represents statisticallyhighly significant differences.)

5. Bacterial Load of Scalded Sites of Rabbits 24 h after Pseudomonasaeruginosa PA14 Infection

The skin and muscular tissue of the scalded and infected sites ofPseudomonas aeruginosa PA14 are taken aseptically, coated to the TSAplates by a homogenizer, and cultured overnight at 37° C.; the CFU iscounted by a colony counter; and then the mean and the standarddeviation of each group are compared based on the log values of thebacterial loads to the base of 10. The results are shown in Table 5 andFIG. 6. The immunization procedure 0, 3, 7 d of the PA1 vaccine cansignificantly reduce the load of Pseudomonas aeruginosa PA14 (P<0.05),with a reduction range of about 0.6-0.9 log. It indicates that thevaccine has a certain protective effect.

It is worth noting that in the experiments for SKLBPA1 and PA14, theimmunization procedure 0, 3, 7 d can quickly generate an effectiveimmune response, so the vaccine of the present invention can be used toprevent skin scald complicated with infection (that is, as aprophylactic vaccine), and may also possibly be used to inhibit andalleviate the infection after the onset of skin scald complicated withinfection (that is, the possibility of serving as a therapeuticvaccine).

TABLE 5 Bacterial Load of Scalded Sites of Rabbits 24 h afterPseudomonas Aeruginosa PA14 infection. Number Bacteria for of SkinsLOG10 of Groups Vaccine Immunization Procedure Infection (blocks)Bacterial Load Group 7 PA1 0, 3, 7 d (injection take places PA14 6  5.67± 0.54* at 0, 3rd, 7th days) Group 8 0, 2, 4 w (injection take places 66.22 ± 1.10 at 0, 2nd, 4th weeks) Group 9 — — 18 6.30 ± 0.65 (Note:*compared with each model group, P < 0.05 represents statisticallysignificant differences.)

The embodiments of the present invention are described above incombination with drawings, but the present invention is not limited tothe aforementioned specific embodiments. The aforementioned embodimentsare merely illustrative and not limiting. For those of ordinary skill inthe art, many forms can be made under the teaching of present inventionwithout departing from the spirit of the present invention and the scopeof the claims, all of which shall fall within the protection scope ofthe present invention.

1. Use of a Pseudomonas aeruginosa vaccine in manufacture of amedicament for prevention and treatment of bacterial infection in burnand scald, wherein the burn and scald are complicated with the bacterialinfection and the bacterial infection is Pseudomonas aeruginosainfection; the Pseudomonas aeruginosa vaccine comprises inactivatedPseudomonas aeruginosa and/or Pseudomonas aeruginosa membrane vesicles;wherein the inactivated Pseudomonas aeruginosa is inactivated byirradiation, and the Pseudomonas aeruginosa membrane vesicles areisolated from the Pseudomonas aeruginosa inactivated by irradiation. 2.The use according to claim 1, wherein the burn and scald include burnsand scalds, and degree of the scalds includes I degree, superficial IIdegree, deep II degree, or III degree scalds.
 3. The use according toclaim 2, wherein site of the scalds includes skin, mucosa or othertissues.
 4. (canceled)
 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. Theuse according to claim 1, wherein site of the scalds includes skin,mucosa or other tissues.
 9. (canceled)
 10. The use according to claim 1,wherein immunization procedures of the Pseudomonas aeruginosa vaccinecomprise: injection take places (i) 0, 3rd, and 7th days, and (ii) 0,2nd and 4th weeks.
 11. (canceled)
 12. The use according to claim 1,wherein the Pseudomonas aeruginosa vaccine prevents Pseudomonasaeruginosa infection, and reduces bacterial load in skin scaldcomplicated with bacterial infection.
 13. The use according to claim 1,wherein content of whole-cell Pseudomonas aeruginosa in the Pseudomonasaeruginosa vaccine comprises: 1×10⁴-1×10¹⁰/injection.
 14. The useaccording to claim 13, wherein the content of whole-cell Pseudomonasaeruginosa in the Pseudomonas aeruginosa vaccine comprises:1×10⁴/injection, 1×10⁵/injection, 1×10⁶/injection, 1×10⁷/injection,1×10⁸/injection, 1×10⁹/injection and 1×10¹⁰/injection.
 15. The useaccording to claim 1, wherein the Pseudomonas aeruginosa vaccine furthercontains an immunoadjuvant.
 16. The use according to claim 15, whereinthe immunoadjuvant is aluminum hydroxide.
 17. The use according to claim1, wherein administration site of the Pseudomonas aeruginosa vaccine issubcutaneous, muscle and/or mucosa.
 18. The use according to claim 1,wherein the medicament can also contain any pharmaceutically acceptablecarrier and/or adjuvant.
 19. The use according to claim 18, wherein thecarrier is a liposome.